During the last several years, significant progress was made in utilization of transgenic plants and other living organisms for production of industrial oils, plastics, edible vaccines and industrially important recombinant proteins. In two latter cases, it was found that plants are able to produce high levels of safe, functional, recombinant proteins and can be easily expanded to agricultural levels to meet industrial demands (Fischer et al., 1999 a, b). Current applications of plants, microorganisms and animal cells in biotechnology include the production of hormones, enzymes, antibodies, plasma proteins, cytokines and vaccines. Recombinant proteins can be produced either by genetically inherited expression in transgenic plants or by transient expression in virus-infected plants.
A revolutionary breakthrough in large-scale production of recombinant proteins in plants was made several years ago by using plant RNA viruses (Kumagai et al., 1993; Hamamoto et al., 1993). The principle of all RNA viral expression systems is the same: upon inoculation, viral RNA replicates in the cytoplasm to high copy number, and the viral progeny RNA is translated resulting in the expression of virally encoded proteins. The virus moves systemically through the whole plant by cell-to-cell and long-distance movement. For vector construction, viral RNA genomes are reverse-transcribed in vitro and cloned as full-length cDNAs in transcription vectors in vitro or in vivo (Boyer, Haenni, 1994). The cloned viral genomes can then be manipulated with standard DNA techniques. For inoculation of plants, recombinant viral vectors are usually transcribed in vitro and the synthesized RNA is inoculated mechanically onto plants by gently rubbing the leaves with a mild abrasive. Extracts from these infected plants can also be used for the subsequent inoculation of very large numbers of plants. One of the most efficient transient expression viral vectors is tobacco mosaic virus (TMV)-based hybrid vectors that contain a heterologous coat protein subgenomic mRNA promoter and coat protein open reading frame and either TMV or heterologous 3′ non-translated region (Shivprasad et al, 1999). The size of the gene that can be expressed with viral vector usually does not exceed 2 kb.
A comparison of features of recombinant protein production in plants, yeast, bacterial, and animal systems is presented in Fischer et al, (1999 a, b). Both transgenic plants and plant viral systems have many advantages compared to the yeast, bacterial, and animal systems. One of the most impressive advantages of the recombinant protein production in plants is the cost of production. The production of proteins from plants infected with viral vectors is several times lower compared to stable transformed transgenic plants. In addition, the time required for the creation of a new plant viral vector product is significantly lower compared to transgenic plants. This low cost and high speed turnaround time are especially important for biotechnological companies. According to data presented by Large Scale Biology (formerly Biosource), the time required to go from a gene expression feasibility study to greenhouse and/or pilot field production, to recovery and purification into purified protein product takes about a year. For transgenic plants it would take at least three years under ideal circumstances. Viral RNA vector systems can be used for the production of different proteins and polypeptides ranging in mass from 4 kd to about 70 kd.
Small epitopic oligopeptides (e.g., less than 25 amino acids in length) can also be produced on the surface of viral particles by gene-fusions created with coat-protein genes (Hamamoto et al., 1993; Fitchen et al., 1995; McLain et al., 1995; Yusibov et al., 1997; Johnson et al., 1997; Koo et al., 1999). There are significant data demonstrating that epitopic oligopeptides on the surface of TMV or other plant viruses induce a strong immune response in vaccinated animals.
On the other hand, for proteins or molecules requiring a high degree of purity, downstream processing from plant biomass is assumed to be generally more problematic and expensive. As a rule, the recombinant product constitutes only a minor fraction of the total biomass. It is well known that in microbial production systems that have been optimized with regard to product yield, up to 90% of total production costs are the costs related to purification of the molecule of interest from the host, rather than expenses of the production itself To make the production from transgenic plants economical, strategies are needed that will allow rapid and inexpensive separation of the recombinant or endogenous proteins of interest or non-proteinaceous small molecules, from other endogenous plant molecules. Since most efficient purification platforms are based on specific affinity between the molecule of interest and the purification matrix, the problem is best addressed by developing a simple and inexpensive high-affinity matrix that can than be used to specifically bind the molecule of interest. One such matrix contemplated in this invention is a protein surface of a virus particle.
The size and biochemical characteristics of every protein are different, so the method of purification must be different for different proteins. One of the approaches that would have a general applicability is the use of separation techniques to concentrate and purify the protein by affinity-mediated isolation. As in microbial systems, expression of the proteins as fusion products (i.e., having an affinity tag) would also facilitate the use of affinity isolation for recovery from plant extracts. There are a number of different commercially available fusion tags for bacterial and animal systems. At least some of them might work in plants; however, they are too expensive for large-scale production.
Ideal characteristics of an ideal affinity tag-based purification system include a homogenous, inexpensive, uniform and specific molecular surface which itself can form large stable aggregates with mass significantly greater than 200-300 kd. This aggregate should bear affinity tags on its surface strong enough to be bound by the protein of interest and to purify it by precipitation. The complex with the protein of interest should in turn, be easy to dissociate under relatively mild ionic conditions. Ideally the affinity matrix would be reusable. Such a system could be used not only in plant biotechnology but also for protein purification in bacterial and animal biotechnology.